CN102512458B - Semi-bionic extraction method for active components of cordyceps militaris - Google Patents

Semi-bionic extraction method for active components of cordyceps militaris Download PDF

Info

Publication number
CN102512458B
CN102512458B CN 201110437505 CN201110437505A CN102512458B CN 102512458 B CN102512458 B CN 102512458B CN 201110437505 CN201110437505 CN 201110437505 CN 201110437505 A CN201110437505 A CN 201110437505A CN 102512458 B CN102512458 B CN 102512458B
Authority
CN
China
Prior art keywords
cordyceps militaris
link
semi
extraction
culture medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN 201110437505
Other languages
Chinese (zh)
Other versions
CN102512458A (en
Inventor
卢丽丽
张丽
陈剑清
舒特俊
张耀洲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guizhou Gui'an precision Medicine Co.,Ltd.
Original Assignee
Is Source Of Tang (tianjin) Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Is Source Of Tang (tianjin) Biotechnology Co Ltd filed Critical Is Source Of Tang (tianjin) Biotechnology Co Ltd
Priority to CN 201110437505 priority Critical patent/CN102512458B/en
Publication of CN102512458A publication Critical patent/CN102512458A/en
Application granted granted Critical
Publication of CN102512458B publication Critical patent/CN102512458B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Abstract

The invention relates to a semi-bionic extraction method for active components of Cordyceps militaris, which belongs to the technical field of processing of biological products. According to the invention, rice and silkworm chrysalis obtained after silk reeling are used as main raw materials to produce Cordyceps militaris, and after low temperature superfine comminution, active components in Cordyceps militaris are extracted in an imitative human gastroenteric acid-base environment by using the semi-bionic extraction method and are optimized. Low temperature superfine comminution in the invention has a good wall breaking effect on spores of Cordyceps militaris, ensures minimum loss of nutrition constituents, enables contents in Cordyceps militaris to be fully exposed and is favorable for implementation of the semi-bionic extraction method. The semi-bionic extraction method simulates human gastroenteric acid-base environment and carries out continuous multiple extraction on the contents of Cordyceps militaris cells, which enables the effect of full extraction to be achieved, ensures that extracted substances are directly absorbed when entering into human gastrointestinal tracts, is beneficial for maximum action of nutrients of Cordyceps militaris in a human body and allows gastrointestinal burden of a human body to be reduced.

Description

The semi-bionic extraction method of Cordyceps militaris (L.) Link. active component
Technical field
The present invention relates to a kind of semi-bionic extraction method of Cordyceps militaris (L.) Link. active component, belong to the biological product processing technique field.
Background technology
Cordyceps militaris (L.) Link. is the fungus take Pupa bombycis as the host, belong to together with Cordyceps is equal, chemical composition is basically identical, has very large medical value, and be the new resource food of Ministry of Public Health approval, have antitumor, antiviral, antibiotic, anti-inflammatory, slow down aging, raising immunity, the multiple efficacies such as blood sugar lowering, applicable crowd is extensive.It is similar with clinical effectiveness to the pharmacological function of Cordyceps, but price is well below Cordyceps, the content of the active component cordycepin that it contains is 10 times in Cordyceps especially, so Cordyceps militaris (L.) Link. becomes the succedaneum of Cordyceps gradually, have great potential market.
The conventional extraction process of Cordyceps militaris (L.) Link., as water extraction, decoction and alcohol sedimentation technique, alcohol extracting methods etc. are keeping effective ingredient, and reject invalid components aspect still exists and extracts fully not, and loss of effective components is large, and operation is many, and the cycle is long, the high in cost of production shortcoming.The Cordyceps militaris (L.) Link. cell wall is hard, and conventional method is difficult to abolish, and brings very large difficulty to leaching process, and causes very large waste and loss.
At present, natural cs output is limited, and quality is uneven and expensive, can't satisfy people's needs, and the Cordyceps militaris (L.) Link. that this method is cultivated is nutritious, quality controllable, and it is low that material damages few cost, and product is easy to preserve, and market potential is huge.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of semi-bionic extraction method of Cordyceps militaris (L.) Link. active component, with by simulation human gastrointestinal tract acid or alkali environment, the Cordyceps militaris (L.) Link. cellular content is carried out continuous several times extract; Than other extracting method, the extraction ratio of resulting active component is high, and quality controllable, and material damages few, and cost is low, is fit to large-scale production, and market potential is huge.
For achieving the above object, the invention provides a kind of semi-bionic extraction method of Cordyceps militaris (L.) Link. active component, said method comprising the steps of:
(1) cultivation of Cordyceps militaris (L.) Link., comprise the steps:
A. the preparation of Chinese caterpillar fungus culture medium: culture medium prescription is dried dried silkworm chrysalis meal 130 ~ 140g/L; Rice meal 60 ~ 65g/L; Potassium dihydrogen phosphate 1.2 ~ 1.5g/L; Sodium dihydrogen phosphate 0.8 ~ 1.2 g/L; Add water and first dissolve KH 2PO 4, NaH 2PO 4, fully add again above-mentioned dried dried silkworm chrysalis meal, rice meal mix homogeneously after mixing, homogenate, packing, every bottled 50 ml build lid, in 121 ℃, moist heat sterilization 30 ~ 45 min under the condition of 1.1Mpa, cooling rear standby;
B. inoculation: inoculate Cordyceps militaris spawn (Cordyceps militaris (L.) Link. is Cordyceps militaris (L.) Link) in step a gained Chinese caterpillar fungus culture medium, its density is 3~5 fungus ball/mL, the volume ratio of described Chinese caterpillar fungus culture medium and described Cordyceps militaris spawn is 50:1, after inoculation under 22 ℃, the condition of humidity 75~85% lucifuge cultivated 3~5 days, make Cordyceps mycelium cover with rapidly media surface, then carry out following illumination cultivation;
The phase I grown cultures: illumination cultivation in trophophase the 5th~20 day, 12 hours daytimes natural lighting, temperature 20 ~ 22 ℃ of (being preferably 22 ℃), humidity 80~85%, intensity of illumination is controlled at 100~150Lx; 12 hours evenings black out, temperature 18 ~ 20 ℃ of (being preferably 18 ℃), humidity 75~85% are ventilated 2 every day, sooner or later each once, ventilated 20~30 minutes at every turn;
The second stage grown cultures: in trophophase the 20th~35 day, 12 hours daytimes, natural light added daylight lamp illumination, temperature 20 ~ 22 ℃ of (being preferably 22 ℃), humidity 75%~85%, and intensity of illumination is controlled at 1000 ~ 1200 Lx; 12 hours evenings black out, temperature 18 ~ 20 ℃ of (being preferably 18 ℃), humidity 75~85% are ventilated 2 every day, sooner or later each once, each ventilation time is 20~30 minutes;
C. the cultivation bottle cap of outwarding winding, tweezers with 75% alcohol disinfecting after, sporophore above step b gained culture medium is taken out, the sporophore that takes out is placed in clean stainless steel disc, select even, sturdy, the complete sporophore of color and luster, remove remaining culture medium be placed on weigh in new stainless steel disc standby; In culture bottle, remainder is cultivated the rear results again that are used for by condition of culture and the method continuation of the 20th ~ 35 day;
D. oven dry: step c gained Cordyceps militaris (L.) Link.sporophore is placed in the stainless steel disc that is lined with gauze, evenly spreads out, 30 ~ 37 ℃ of hot air circulation oven dry are stand-by, and moisture finally is controlled at below 10%;
(2) superfine comminution at low temperature: take the Cordyceps militaris (L.) Link.sporophore of 200 ~ 250 g oven dry at every turn, put into super micron mill and pulverize 5 ~ 7 min, during pulverizing with the water of 4 ~ 10 ℃ as liquid coolant, reduce and pulverize temperature, protect the active component in Cordyceps militaris (L.) Link.;
(3) semi-bionic extraction of Cordyceps militaris (L.) Link. active component: the acid water (pH value is 1 ~ 6) that adopts selected pH value, neutral water (pH value is 6 ~ 8) and alkaline water (pH value is 8 ~ 14), and selected time (total extraction time 50 ~ 180min), be under 1 ~ 6 sour environment, Cordyceps militaris (L.) Link. to be extracted prior to pH value, collect supernatant with the centrifugal force of 8000 ~ 10000g after centrifugal 5 ~ 10 minutes, be to extract under 6 ~ 8 neutral environments in pH value again after the precipitation natural air drying, collect supernatant with same centrifugal force after centrifugal 5 ~ 10 minutes, be to extract in 8 ~ 14 alkaline environments in pH value at last, by the centrifugal collection supernatant of above-mentioned same condition, each solid-liquid ratio that extracts solvent for use water and crude drug is 5 ~ 15 times, and the solid-liquid ratio of three extractions can be different, merge three times supernatant, be semi-biomimetic method and extract the Cordyceps militaris (L.) Link. active component that obtains.
Preferably, also be added with pepsin, trypsin, alpha-glucosidase, chymase, elastoser, erepsin or lipase in wherein said step (3).
The present invention has following beneficial effect:
1. adopt the semi-bionic extraction method, simulation human gastrointestinal tract acid or alkali environment carries out the extraction of Cordyceps militaris (L.) Link. active component and it is optimized, and the environment that soda acid changes is conducive to increase the stripping of active component in medicine, makes extraction more abundant.And avoided extracting with hazardous solvent, gains are safe and effective;
2. the use super micron mill, carry out superfine comminution at low temperature, to abolish the hard cell wall of Cordyceps militaris (L.) Link.sporophore, is conducive to the stripping of content, improves the extraction ratio of active component in Cordyceps militaris (L.) Link.;
3. adopt the active component in freeze-drying concentrate drying Cordyceps militaris (L.) Link., the method not only can guarantee the good water-soluble effect of the final active component that obtains, and carry out drying under the condition of cryogenic vacuum, and can effectively guarantee the activity of extract in Cordyceps militaris (L.) Link., guarantee its medical value;
4. the method extraction Cordyceps militaris (L.) Link. nutrition content is high, take the quality of the cordycepin that extracted and final gained active substance as standard, obtain the optimal conditions of semi-bionic extraction process, under this condition, the content of cordycepin can reach every g crude drug and extract the amount that obtains 7.417mg, and the extraction ratio of final active component can be up to 79.54%; Than other extracting method, the extraction ratio of resulting active component is all high, and quality controllable, and material damages few, and cost is low, is fit to large-scale production, and market potential is huge;
5. simulation human gastrointestinal tract acid or alkali environment carries out the extraction of Cordyceps militaris (L.) Link. active component, after production and processing becomes the dosage form of oral liquid or other types, can guarantee the direct absorption of Cordyceps militaris (L.) Link. effective ingredient, alleviate the intestines and stomach burden, and give full play to the important function of this precious Chinese medicine of Cordyceps militaris (L.) Link..
Description of drawings
Fig. 1 is the scanning electron microscope (SEM) photograph after the broken Cordyceps militaris (L.) Link. of the common breaking method of employing;
Fig. 2 is the scanning electron microscope (SEM) photograph after the ultra micro pulverize at low temperature;
Fig. 3 is the canonical plotting of cordycepin.
The specific embodiment
Be noted that following illustrating is all exemplary, be intended to the invention provides further instruction.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of usually understanding with the technical field of the invention personnel.
The Pupa bombycis of method of the present invention after the filature is as primary raw material, and rice is the cultivation that adjuvant carries out Cordyceps militaris (L.) Link..Its nutrient composition content is far above the component content in the Cordyceps of adopting additive method to cultivate, and color is golden yellow glossy, suffuses an exquisite fragrance all around, and adenosine and cordycepin content are higher than natural Cordyceps; Carrying out multiple cold sterilization after the results oven dry processes, adopt superfine comminution at low temperature to reduce the loss of nutritional labeling in the shell-broken effect that guarantees the Cordyceps militaris (L.) Link. spore, the aqueous solution that constantly changes with acid or alkali environment carries out the extraction of Cordyceps militaris (L.) Link. active substance under low temperature, be not destroyed to guarantee active component.Carry out the extraction of Cordyceps militaris (L.) Link. active component by simulation human gastrointestinal tract acid or alkali environment, after production and processing becomes drug formulation, can guarantee the direct absorption of Cordyceps militaris (L.) Link. effective ingredient, alleviate the intestines and stomach burden, and give full play to the important function of this precious Chinese medicine of Cordyceps militaris (L.) Link..
Elaborate particular content of the present invention below in conjunction with embodiment.
Embodiment 1: the cultivation of Cordyceps militaris (L.) Link.
A. the configuration of culture medium: Chinese caterpillar fungus culture medium of the present invention is aqueous solution, is made of the composition of following concentration:
Dried dried silkworm chrysalis meal 140 g/L;
Rice meal 60 g/L;
Potassium dihydrogen phosphate 1.2 g/L;
Sodium dihydrogen phosphate 1.2 g/L;
Preferred concrete preparation method is as follows:
Accurately take dried dried silkworm chrysalis meal 2.1kg, rice meal 900g, potassium dihydrogen phosphate 18g after sodium dihydrogen phosphate 18g, adds pure water 12 L, first dissolves KH 2PO 4, NaH 2PO 4, fully obtain the culture medium solution that volume is 15L after mixing, then add above-mentioned dried dried silkworm chrysalis meal, rice meal mix homogeneously, homogenate, packing, every bottled 50 ml build lid, in 121 ℃, moist heat sterilization 30 min under the condition of 1.1Mpa, cooling rear standby.
B. inoculation: (strain comes from Suzhou Silkworm Training School to strain to inoculate Cordyceps militaris (L.) Link. (Cordyceps militaris (L) Link) in super-clean bench, tribute professor Cheng Liang give), liquid spawn density is controlled at 3-5 fungus ball/mL, every bottle graft kind 1 mL, after inoculation under 22 ℃, the condition of humidity 75~85% lucifuge cultivated 3~5 days, make Cordyceps mycelium cover with rapidly media surface, then carry out following illumination cultivation;
The phase I grown cultures: cultivate illumination in the 5th~20 day, illumination on 12 hours daytimes (getting final product with natural light), 22 ℃ of temperature, humidity 80~85%, intensity of illumination is controlled at 100~150Lx; 12 hours evenings black out, 18 ℃ of temperature, humidity 75~85%.Ventilate every day 2 times, each once, ventilated 20~30 minutes at every turn sooner or later;
The second stage grown cultures: illumination on 12 hours daytimes (natural light adds daylight lamp) in the 20th~35 day, 22 ℃ of temperature, humidity 75%~85%, intensity of illumination is controlled at 1000 Lx; 12 hours evenings black out, 18 ℃ of temperature, humidity 75~85% are ventilated 2 every day, sooner or later each once, each ventilation time is 20~30 minutes.
Points for attention:
1. will use scattered light during illumination, and can not use direct light, the growth of Cordyceps has phototropism, be in illumination uniformly local bottle to note rolling bottle, to promote upwards growth of sporophore;
2. to strictly control temperature and humidity in incubation.If the workshop does not have attemperating unit, can control by the mode of sprinkling water in ground the temperature of culturing room.The temperature of culturing room can not be over 25 ℃;
3. note number of times and the time of ventilation;
4. the bottle that pollutes will in time be taken out processing, with the expansion of avoiding polluting.
C. results and cultivation again: the cultivation bottle cap of outwarding winding, tweezers with 75% alcohol disinfecting after, sporophore is taken out (only taking out the part above culture medium), the sporophore that takes out is placed in clean stainless steel disc, select even, sturdy, the complete sporophore of color and luster, remove remaining culture medium be placed on weigh in new stainless steel disc standby; In culture bottle, remainder can be gathered in the crops after continuing to cultivate by the condition of culture of the 20th ~ 35 day and method again.
D. oven dry: Cordyceps militaris (L.) Link.sporophore is placed in the stainless steel disc that is lined with gauze, evenly spreads out, 37 ℃ of hot air circulation oven dry are stand-by, and moisture finally is controlled at below 10%.
Embodiment 2: superfine comminution at low temperature
Take the Cordyceps militaris (L.) Link.sporophore of 200 g oven dry at every turn, put into super micron mill and pulverize 6 min, during pulverizing with the water of 4 ~ 10 ℃ as liquid coolant, reduce and pulverize temperature, protect the active component in Cordyceps militaris (L.) Link..After pulverizing, sampling detects shell-broken effect with scanning electron microscope.Effect after common fragmentation and micronized pulverization is seen respectively Fig. 1 and Fig. 2.By comparison diagram 1 and Fig. 2 as seen, after common fragmentation, the cell wall of Cordyceps militaris (L.) Link.sporophore is complete, and cell still becomes agglomerate to assemble.Cell wall after the micronizing fragmentation is imperfect, and content mays be seen indistinctly, therefore the Cordyceps militaris (L.) Link. spore after micronizing is thoroughly broken, nutritional labeling has obtained effective release.
Embodiment 3: the semi-bionic extraction of Cordyceps militaris (L.) Link. active component
by imitating the transport process of oral drugs in gastrointestinal tract, adopt the acid water (pH value is 1 ~ 6) of selected pH value, neutral water (pH value is 6 ~ 8) and alkaline water (pH value is 8 ~ 14), and selected time (total extraction time 50 ~ 180min), be under 1 ~ 6 sour environment, Cordyceps militaris (L.) Link. to be extracted prior to pH value, collect supernatant with the centrifugal force of 10000g after centrifugal 10 minutes, under being 6 ~ 8 neutral environment, extracts pH value again after the precipitation natural air drying, collect supernatant with same centrifugal force after centrifugal 10 minutes, in being 8 ~ 14 alkaline environment, extracts pH value at last, by the centrifugal collection supernatant of above-mentioned same condition.Each solid-liquid ratio that extracts solvent for use water and crude drug is 5 ~ 15 times, and the solid-liquid ratio of three extractions can be different.Merge three times supernatant, this is semi-biomimetic method and extracts the Cordyceps militaris (L.) Link. active component that obtains.
Get 100 μ l extracting solution and be diluted to the filter membrane of crossing 0.22 μ m after 1000 μ l, measure cordycepin content in high performance liquid chromatograph, the lyophilizing in freezer dryer of remaining extracting solution.This step can be optimized extraction scheme by uniform design or orthogonal experiment, and the factor of consideration desolventizes outside pH, also can consider extraction time, temperature, and the factors such as extraction water consumption are optimized extraction conditions as far as possible.For strengthening mass transport process, except using ultrasonic extraction, also can use microwave extraction in leaching process, the modes such as concussion extraction are strengthened mass transport process, and the simulation gastrointestinal peristalsis with the stripping of more effective promotion cellular content, is strengthened leaching process.The semi-bionic extraction process also can add pepsin, trypsin, the enzyme such as alpha-glucosidase, chymase, elastoser, erepsin, lipase reach bionic extraction as far as possible, finally reach the more abundant active component of extracting more efficiently in Cordyceps militaris (L.) Link..
Below by different time and the pH value of design, Cordyceps militaris (L.) Link. is carried out 3 water extractions and optimization, the quality of the dry of gained is estimated the quality of extraction effect as standard after the cordycepin content that extracted and the lyophilization.Cordycepin content is measured with high performance liquid chromatograph.
The uniform design Optimized Extraction Process is adopted in this experiment, and the uniform Design factor is four factors, and level is six levels.Ultrasonic extraction acid water pH value used is respectively 1.00,2.00,3.00 for the first time, 4.00,5.00 and 6.00, the pH value of ultrasound wave water extraction neutral water used is respectively 6.50 for the second time, 7.00 with 7.50, the pH value of the alkaline water of ultrasound wave water extraction is 8.00,9.00 and 10.00 for the third time.Extraction was respectively 55 minutes total time, and 77 minutes and 99 minutes, three times are extracted time scale used was 5:3:3.Use the experiment of uniform design reasonable arrangement to layout.The solid-liquid ratio that extracts for three times is respectively 10,8,8.Each extract finish after in the centrifugal force of 10000g, under the condition of 4 ℃ centrifugal 10 minutes.
Be to guarantee comparability and the repeatability of result, in the decoction pieces specification, extract temperature, extract water consumption, under prerequisite that filtration, the condition such as concentrated are identical, determine principal element and the level investigated, content sees the following form 1:
Table 1:
Figure 800762DEST_PATH_IMAGE001
Wherein, 3 times extraction time ratio be 5:3:3.
Adopt uniform design optimization experiment scheme, uniform designs table U6* (64) arranges to layout to see the following form 2:
Table 2:
Figure DEST_PATH_IMAGE002
(1) chromatographic condition:
Apollo C18 post (250 mm * 4.6 mm, 5 μ m), 30 ℃ of column temperatures, detect wavelength 260nm, mobile phase methanol (chromatographic grade 100%)-0.01mol/L sodium phosphate buffer (pH6.6) 15:85, flow velocity 0.700ml/min, sample size 10 μ l.
(2) preparation of sample solution:
Take Cordyceps militaris (L.) Link. micronizing powder 0.5000 gram (every group of positive negative error is no more than 0.0005 gram), according to upper surface condition water-bath supersound extraction, the ultrasonic extraction temperature is 20 ℃, each extract finish after in centrifuge with the centrifugal force of 10000g centrifugal 10 minutes, collect to merge the water system filter membrane of crossing 0.45 μ m after supernatant, be settled to get after 25ml and measure cordycepin content after 100 μ l are diluted to ultra-pure water the water system filter membrane that 1000 μ l cross 0.22 μ m in high performance liquid chromatograph.
(3) preparation of standard solution:
The purity that is purchased from the cordycepin standard substance of sigma company is 99.9%, and the cordycepin standard substance of 10mg are dissolved in 5ml, in 100% hplc grade methanol, is prepared into the storing solution of 2mg/ml.
Get the standard stock solution of 8 μ l, be diluted to the standard solution of 40 μ g/ml with ultra-pure water, and then be diluted to successively 20 μ g/ml, 5 μ g/ml, the standard solution of 1 μ g/ml.The standard solution test of each concentration 3 times.According to (1) chromatographic condition sample introduction, with peak area Y, concentration X (μ g/ml) is done standard curve, get equation of linear regression.
(4) mensuration of instrument precision:
Get the standard solution of 5 μ g/ml by (1) condition continuous sample introduction 5 pins, the precision result is with relative standard deviation RSD value representation.
(5) response rate experiment
Get the standard substance (low) that No. 1 sample adds respectively 4 μ g, the standard substance of 8 μ g (in), the standard substance of 12 μ g (height), each sample continuous sample introduction 3 pin, calculate recovery rate and relative standard variance RSD value.
3, optimize the extraction result
(1) instrument precision measurement result 3 (with standard substance continuous sample introduction 5 pins of 5 μ g/ml) that see the following form.
Table 3:
Figure 717903DEST_PATH_IMAGE003
As shown in Table 3: relative standard variance RSD value<15% illustrates that instrument precision is good.
(2) cordycepin standard substance measurement result sees the following form 4.
Table 4:
Standard substance concentration/μ g/ml Peak area
1 47850
5 258817
20 1112332
40 2211727
As shown in table 4: as with peak area Y, concentration X (μ g/ml) to be done standard curve (standard curve is seen Fig. 3), get equation of linear regression: y=55657x-10660 (R 2=0.9999), relative standard variance R 2Be 0.9999, the description standard curve linear is on good terms, and institute's measured value is true and reliable, and standard curve can be used.
(3) sample determination the results are shown in following table 5.
Table 5:
Sequence number Peak area Cordycepin concentration/μ g/ml Cordycepin content in every g crude drug/μ g/g
1 706931 12.893 6446.552
2 815002 14.835 7417.415
3 507134 9.303 4651.649
4 574334 10.511 5255.354
5 656451 11.986 5993.053
6 639898 11.689 5844.347
As shown in Table 5: extracting optimum condition is the 2nd set condition: namely the ultrasonic extraction pH value is 2.00 for the first time, and the ultrasonic extraction pH value is 7.00 for the second time, and the ultrasonic extraction pH value is 10.00 for the third time.The ultrasonic extraction time is 45 minutes for supersound extraction time for the first time, and the ultrasonic extraction condition is 27 minutes for the second time, and the supersound extraction condition is 27 minutes for the third time, and total time is 99 minutes.With this understanding, gained cordycepin extraction ratio is can extract the cordycepin of 7.417mg in every g raw medicinal herbs.
(4) response rate experiment sees the following form 6.
Table 6:
Background concentration/μ g Addition/μ g Measured value The response rate/% Meansigma methods RSD/%
4 (low) 915329 93.611
12.893 4 (low) 914975 93.452 93.353 0.342
4 (low) 913960 92.996
8 (in) 905392 89.147
12.893 8 (in) 905690 89.281 89.206 0.077
8 (in) 905489 89.191
12 (height) 863036 70.122
12.893 12 (height) 863235 70.211 70.144 0.084
12 (height) 862988 70.100
As shown in Table 6, the response rate reaches more than 70%, illustrates that the accuracy of assay method is higher.
4. dry matter content
With extract lyophilizing in freezer dryer, the quality of last gained dry sees the following form 7.
Table 7:
Sequence number Cordycepin content in every g crude drug/μ g/g Quality/the g of dry Active component extraction ratio/%
1 6446.552 0.3092 61.84
2 7417.415 0.3977 79.54
3 4651.649 0.2741 54.82
4 5255.354 0.3361 67.22
5 5993.053 0.3679 73.58
6 5844.347 0.3528 70.56
As shown in Table 7, extract optimum condition and be still second group, carrying out semi-bionic extraction with this condition can obtain its active component extraction ratio and can reach 79.54%, the active component that will obtain than general extracting method is all high, and adopt freeze-drying to carry out concentrate drying to active component, can farthest keep the activity of material, guarantee its drug effect.
The above, be only the preferred embodiments of the present invention, should be understood that, for the those of ordinary skill in present technique, under the future that does not break away from core technology of the present invention, can also make and improving and retouching, as consider extraction time, add enzyme such as pepsin in gastrointestinal tract in leaching process, trypsin etc. make the semi-bionic extraction technology as far as possible near real human gastrointestinal tract environment, finally reach the effect of real bionic extraction, precious composition in Cordyceps militaris (L.) Link. is fully dissolved, cut the waste simultaneously, reduce costs; These retouchings and improvement also should belong to scope of patent protection of the present invention.

Claims (5)

1. the semi-bionic extraction method of a Cordyceps militaris (L.) Link. active component, is characterized in that, said method comprising the steps of:
(1) cultivation of Cordyceps militaris (L.) Link.: take Pupa bombycis as primary raw material, rice is adjuvant, adds phosphate and water to be mixed with culture medium Cordyceps militaris (L.) Link. is cultivated;
(2) superfine comminution at low temperature: abolish the hard cell wall of Cordyceps militaris (L.) Link.sporophore by the superfine comminution at low temperature technology;
(3) semi-bionic extraction of Cordyceps militaris (L.) Link. active component: the employing pH value is 1 ~ 6 acid water, pH value is that 6 ~ 8 neutral water and pH value are 8 ~ 14 alkaline water, and total extraction time 50 ~ 180min, be under 1 ~ 6 sour environment, Cordyceps militaris (L.) Link. to be extracted prior to pH value, collect supernatant with the centrifugal force of 8000g ~ 10000g after centrifugal 5 ~ 10 minutes, be to extract under 6 ~ 8 neutral environments in pH value again after the precipitation natural air drying, collect supernatant with same centrifugal force after centrifugal 5 ~ 10 minutes, be to extract in 8 ~ 14 alkaline environments in pH value at last, by the centrifugal collection supernatant of above-mentioned same condition, each solid-liquid ratio that extracts solvent for use water and crude drug is 5 ~ 15 times, and the solid-liquid ratio of three extractions can be different, merge three times supernatant, be semi-biomimetic method and extract the Cordyceps militaris (L.) Link. active component that obtains.
2. method according to claim 1, is characterized in that, described step (1) comprising:
A. the preparation of Chinese caterpillar fungus culture medium: culture medium prescription is dried dried silkworm chrysalis meal 130 ~ 140 g/L; Rice meal 60 ~ 65 g/L; Potassium dihydrogen phosphate 1.2 ~ 1.5g/L; Sodium dihydrogen phosphate 0.8 ~ 1.2 g/L; Add water and first dissolve KH 2PO 4, NaH 2PO 4, fully add again above-mentioned dried dried silkworm chrysalis meal, rice meal mix homogeneously after mixing, homogenate, packing, every bottled 50 ml build lid, in 121 ℃, moist heat sterilization 30 ~ 45 min under the condition of 1.1Mpa, cooling rear standby;
B. inoculation: inoculate Cordyceps militaris spawn in step a gained Chinese caterpillar fungus culture medium, its density is 3~5 fungus ball/mL, the volume ratio of described Chinese caterpillar fungus culture medium and described Cordyceps militaris spawn is 50:1, after inoculation under 22 ℃, the condition of humidity 75~85% lucifuge cultivated 3~5 days, make Cordyceps mycelium cover with rapidly media surface, then carry out following illumination cultivation;
The phase I grown cultures: illumination cultivation in trophophase the 5th~20 day, 12 hours daytimes natural lighting, 20 ~ 22 ℃ of temperature, humidity 80~85%, intensity of illumination is controlled at 100~150Lx; 12 hours evenings black out, 18 ~ 20 ℃ of temperature, humidity 75~85% are ventilated 2 every day, sooner or later each once, ventilated 20~30 minutes at every turn;
The second stage grown cultures: in trophophase the 20th~35 day, 12 hours daytimes, natural light added daylight lamp illumination, 20 ~ 22 ℃ of temperature, humidity 75%~85%, and intensity of illumination is controlled at 1000 ~ 1200 Lx; 12 hours evenings black out, 18 ~ 20 ℃ of temperature, humidity 75~85% are ventilated 2 every day, sooner or later each once, each ventilation time is 20~30 minutes;
C. results and cultivation again: the cultivation bottle cap of outwarding winding, tweezers with 75% alcohol disinfecting after, sporophore above step b gained culture medium is taken out, the sporophore that takes out is placed in clean stainless steel disc, select even, sturdy, the complete sporophore of color and luster, remove remaining culture medium be placed on weigh in new stainless steel disc standby; In culture bottle, remainder is cultivated the rear results again that are used for by condition of culture and the method continuation of the 20th ~ 35 day;
D. oven dry: step c gained Cordyceps militaris (L.) Link.sporophore is placed in the stainless steel disc that is lined with gauze, evenly spreads out, 30 ~ 37 ℃ of hot air circulation oven dry are stand-by, and moisture finally is controlled at below 10%.
3. method according to claim 2, is characterized in that, the Cordyceps militaris (L.) Link. in described step b is Cordyceps militaris (L.) Link., and Cordyceps militaris spawn is the Cordyceps militaris strain.
4. method according to claim 2, is characterized in that, described step (2) comprising:
Take the Cordyceps militaris (L.) Link.sporophore of 200 ~ 250 g oven dry at every turn, put into super micron mill and pulverize 5 ~ 7 min, during pulverizing with the water of 4 ~ 10 ℃ as liquid coolant, pulverize temperature to reduce, protect the active component in Cordyceps militaris (L.) Link..
5. method according to claim 1, is characterized in that, described step also is added with pepsin, trypsin, alpha-glucosidase, chymase, elastoser, erepsin or lipase in (3).
CN 201110437505 2011-12-23 2011-12-23 Semi-bionic extraction method for active components of cordyceps militaris Active CN102512458B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201110437505 CN102512458B (en) 2011-12-23 2011-12-23 Semi-bionic extraction method for active components of cordyceps militaris

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110437505 CN102512458B (en) 2011-12-23 2011-12-23 Semi-bionic extraction method for active components of cordyceps militaris

Publications (2)

Publication Number Publication Date
CN102512458A CN102512458A (en) 2012-06-27
CN102512458B true CN102512458B (en) 2013-05-22

Family

ID=46283712

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110437505 Active CN102512458B (en) 2011-12-23 2011-12-23 Semi-bionic extraction method for active components of cordyceps militaris

Country Status (1)

Country Link
CN (1) CN102512458B (en)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103141298B (en) * 2013-02-05 2014-05-14 浙江省林业科学研究院 Fermentation and extraction method for active components in cordyceps sinensis mycelium
CN103271952A (en) * 2013-06-07 2013-09-04 陈玉龙 Preparation method and preparation of cordyceps sinensis active concentration powder
CN103710241B (en) * 2013-12-20 2016-05-04 正源堂(天津)生物科技有限公司 A kind of preparation method of Cordyceps militaris assembled alcoholic drinks
CN103740556A (en) * 2013-12-20 2014-04-23 正源堂(天津)生物科技有限公司 Preparation method of dry cordyceps militaris compound wine
CN103766914B (en) * 2014-01-24 2016-02-17 侯梦斌 A kind of semi-bionic extraction method method of dietary supplements
CN104585733B (en) * 2014-12-31 2016-08-24 台州康晶生物科技有限公司 Cordyceps oral tablet and preparation method thereof
CN104705642A (en) * 2014-12-31 2015-06-17 浙江理工大学 Cordyceps militaris oral tablet and preparation method thereof
WO2017084030A1 (en) * 2015-11-17 2017-05-26 罗明镜 Method for extracting cordyceps sinensis extract
WO2017084029A1 (en) * 2015-11-17 2017-05-26 罗明镜 Method for extracting cordyceps sinensis extract
CN106434806B (en) * 2016-09-30 2019-09-03 北华大学 Cordyceps militaris polypeptide and its preparation method and application
CN113388045A (en) * 2020-03-13 2021-09-14 宁波易中禾药用植物研究院有限公司 Extraction method of dendrobium officinale with antibacterial effect and compound killing preparation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173218A (en) * 2007-09-30 2008-05-07 袁有宝 Cultivate method for cordyceps militaris link

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173218A (en) * 2007-09-30 2008-05-07 袁有宝 Cultivate method for cordyceps militaris link

Also Published As

Publication number Publication date
CN102512458A (en) 2012-06-27

Similar Documents

Publication Publication Date Title
CN102512458B (en) Semi-bionic extraction method for active components of cordyceps militaris
CN102558265B (en) Extract method of cordyceps militaris adenosine
Zhou et al. Applied modern biotechnology for cultivation of Ganoderma and development of their products
CN102515920B (en) Culture of cordyceps militaris and preparation method of oral cordyceps militaris tablet
CN104013657B (en) A kind of American ginseng medicine extracts after saponin(e microbial fermentation extracting method again
CN101095710B (en) Method for improving the main efficacy composition of panax notoginseng through zymolysis
CN102242069B (en) Paecilomycescicadae (Miq.)Samson and application thereof
CN104623122B (en) A kind of biological agent and preparation method thereof with anti-fatigue effect
CN105193876A (en) Purslane extract and preparation method thereof
Tao et al. Optimization of the Artemisia polysaccharide fermentation process by Aspergillus niger
CN108713447A (en) A kind of high altitude localities local tyrant meat Mythic Fungus cultivation method and its health products preparation method
CN106171521B (en) A kind of cultural method of rose agaric culture material and its rose agaric
CN106072632B (en) Ganoderma lucidum extract and preparation method and application thereof
CN1005525B (en) Rapid solid culture of edible fungus mycelium for food processing
CN109200082A (en) A method of extracting from emblic, there is antioxidant activity to have Substance simultaneously
CN110079482A (en) A kind of feeding bacillus amyloliquefaciens and its application
CN105769938B (en) One plant of light brown Aode mushroom and its application
CN104903454A (en) Process for the large scale production of fruit cells
CN109223835A (en) A kind of method of cordyceps sinensis Relative Fungi common fermentation production Radix Astragali Radix Notoginseng mycoplasma
CN104254594A (en) Functional makgeolli with improved radioactive strontium discharge and antioxidant activity, containing selenium solution, and preparation method thereof
CN101245334A (en) Technique for suspension cultivation of algam dendrobium nobile embryoid of medicinal effective composition of native plant strain
CN107550945A (en) Protect Pleurotus citrinopileatus extract and its application of wounded hepatocytes
CN106822597A (en) A kind of novel fermentation Chinese medicine and its production and use
CN105560608A (en) Pharmaceutical composition for treating lung cancer
CN104082134A (en) Dendrobium embryoid suspension culture process

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
ASS Succession or assignment of patent right

Owner name: ZHANG YAOZHOU

Free format text: FORMER OWNER: ZHENGYUANTANG (TIANJIN) BIOTECHNOLOGY CO., LTD.

Effective date: 20140626

C41 Transfer of patent application or patent right or utility model
COR Change of bibliographic data

Free format text: CORRECT: ADDRESS; FROM: 300457 BINHAI NEW DISTRICT, TIANJIN TO: 310000 HANGZHOU, ZHEJIANG PROVINCE

TR01 Transfer of patent right

Effective date of registration: 20140626

Address after: 310000 Jianggan District, Zhejiang, No. 2, No. street, No. 5

Patentee after: Zhang Yaozhou

Address before: 300457 Tianjin Binhai New Area Economic Development Zone Dongting Road No. 220 Tianjin Institute of international medicine, building 5, N511

Patentee before: Is the source of Tang (Tianjin) Biotechnology Co. Ltd.

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20170413

Address after: 550025 Guizhou Province in the New District of Guizhou security comprehensive security zone

Patentee after: Guizhou Institute of precision medicine Limited by Share Ltd

Address before: 310000 Jianggan District, Zhejiang, No. 2, No. street, No. 5

Patentee before: Zhang Yaozhou

CP01 Change in the name or title of a patent holder

Address after: 550025 Guian Comprehensive Bonded Zone, Qianzhong Road, Guian New District, Guizhou Province

Patentee after: Guizhou Gui'an precision Medicine Co.,Ltd.

Address before: 550025 Guian Comprehensive Bonded Zone, Qianzhong Road, Guian New District, Guizhou Province

Patentee before: ACADEMY OF PRECISION MEDICINE, GUIAN, GUIZHOU Co.,Ltd.

CP01 Change in the name or title of a patent holder